Notes BIO325 - Genetics
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This 45 page Class Notes was uploaded by Ria Cho on Friday February 6, 2015. The Class Notes belongs to BIO325 - Genetics at University of Texas at Austin taught by Dr. Finklea in Fall. Since its upload, it has received 107 views. For similar materials see Genetics in Biology at University of Texas at Austin.
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Date Created: 02/06/15
DNA Structure 09112014 Experiments in the 1920 s40 s Revealed true nature of DNA 0 1 in uences traits phenotypes 0 Originally thought to be protein 0 Genetic material is inherited physical molecule 0 2 Parentljoffspring All cells have DNA as genetic material 0 Including prokaryotes RNA is the genetic material of some viruses 0 Can also be DNA 0 Some have multiple chromosomes DNA RNA Double stranded Single stranded Common difference Deoxyribose no oxygen Ribose oxygen De ning difference ATCG bases AUCG bases Common difference Numbering carbons Exocyclic carbon is 5 0 Carbon that determines whether it is DNA or RNA is 2 Purines 0 AC o Aggies eat Purina Double ring Pyrimidines T U C 0 Single ring bases attached to 1 C phosphatases 13 attached in series to 5 C Naming nucleotides 1 Identify sugar things can turn around 2 Identify base 3 Name phosphates ex dADP deoxyribose adenine diphosphate dGMP deoxyribose guanine monophosphate ATP ribose adenine triphosphate Chargaff s Rule Analyzed DNA from all different species 0 Looking for content of different bases relative to each other No speci c for each base 0 A T o C G Rosalind Franklin Xray crystallographydiffraction o Able to predict dimensions of DNA Very dif cult to do Technique worked and still used today Watson amp Crick Versed in chemistry and physics 0 Used data of Charga and Franklin 0 Made models to nd what t with that data 0 2 strands are antiparallel opposite directions applies to any double stranded nucleic acid even RNA n mRNA pairs with tRNA antiparallel so polarity 5 and 3 matches up 0 bases on the inside connect to bases on other strand complementary base pairing AT GC Chargaff s rule amp Franklin s dimensions and molecules data based Watson and Crick s base pairing rule Electronegativity o Atoms with high EN hog time w e O N Atoms with low EN have less time w e C H Major and minor grooves 2 sugar phosphate backbones grooves don t intersect o wider is major narrower is minor bases in the grooves 0 access to bases does not always need strand to pull apart 0 proteins don t recognize backbone because it s all the same 0 instead they bind to the grooves amp speci c bases DNA binding domains appendages of proteins a Fit in either top of major or bottom of minor groove 0 AT reads different from TA when read from top of major groove because of speci c pattern of partial charges 0 More limited in minor grooves More proteins bind to the major groove Forms of DNA 0 Proteins can bind to speci c sequences on DNA 0 H bonds on R groups 0 Nonpolar interactions with neutral points Nonpolar amino acids depending on folding of polypeptide Semiconservative ALWAYS New strands contain one old strand and one new strand Conservative NEVER One daughter molecule is the original 0 Other daughter has 2 new strands Strands must separate 1 unwind o breaking of H bonds 2 melt o boilingnear boiling to break H bonds 3 denature 0 use different chemicals to destabilize H bonds Renatu ration Removing whatever it was that separated the strands and broke H bonds DNA will renature on its own as long as complementary bases are present 0 Double strandedower energy state E Coli base for prokaryotic replication Not universal but representative Replication begins at the origin of replication instead of everywhere on the strand all at the same time DnaA Box binding site DnaA protein that binds at site 0 Bind to each other without releasing DNA DNA must bend a Slight unwinding forcepressure on the strand 0 not enough force on an average piece of DNA only 2 H bonds per base pair in AT rich regions vs 3 bonds in GC rich regions 0 not as tightly bound easily meltable bending is effective 0 AT rich regions and DnaA box cannot generate origins of replication individually 0 however when DnaA box causes bending near an AT rich regionorigin of replication is formed HeHcase Enzyme that breaks H bonds and unzips strands 0 Multiple proteins come and bind at forks 0 Work going outwards 0 Larger and larger replication bubble Side twists are called supercoils 0 Blocks any more unwinding Topoisomerase 0 Type 1 at the heads of the fork o Breaks phosphodiester bonds in DNA One of the backbones o Takes out a twist Can rotate broken strand around unbroken strand n Reforms phosphodiester bond 0 Moves along ahead of the fork and continues 0 Type 2 breaks both strands o Untwists both amp reconnects strands and phosphodiester bonds DNA Polymerase 3 Replicates most of the DNA in prokaryotes DNA Polymerase 1 o Removes primers and lls in the gaps with DNA in prokaryotes Eukaryotes Linear chromosomes 0 Multiple origins of replication Separate parts of the chromosome replicate simultaneously a Speeds up replication Eukaryotes are much larger and take much more time to replicate o Prokaryotes take 2030 min to complete replication 0 Eukaryotes take 8 hours to complete replication 0 Bidirectional Primers are removed and fragments are connected when forks run into each other 0 New nucleotides must be 0 Complementary to template nucleotides 0 ln triphosphate form DNA Polymerase will break the bond between 2 of the phosphates and the remaining phosphate on the nucleotide Each nucleotide is broken down to power its own addition to the backbone o Phosphate must be bonded between phosphate 5 C to OH 3 C DNA Polymerase can only add nucleotides at the 3 end 0 Needs a primer with a 3 end 0 Until new strand exists there is no 3 end 0 Primer made out of RNA Enzymes are processive 0 When without dissociating it repeats enzymatic activity 0 High if thousands of times 0 Low if tens of times DNA Polymerase d alpha 5 3 DNA Polymerase activity makes DNA 5 3 RNA Polymerase activity makes RNA 0 2 active sites makes short primer on one site roll over and uses other site 0 does not proofread 0 higher errormutation rate 0 lower processivity 0 not a limitation DNA Polymerase Z epsilon amp DNA Polymerase 6 delta 0 High processivity o Accurate and ef cient 5 3 DNA Polymerase activity 0 triphosphate nucleotides pairs up with template polymerase removes 2 phosphates and uses energy checks most recent pair made by checking helix shape 3 5 exonuclease activity 0 if distortion is present DNA pol backs up 3 5 breaks phosphodiester bond n exonuclease backing up and removing one nucleotide to proofread 0 high processivity 0 has clamp attached to it forward to back motion not limited dissociation hindered makes most of DNA DNA Pol Z is the major replication enzyme on the leading strand DNA Pol 6 is the major replication enzyme on the lagging strand Leading strand 0 Continuous replication Synthesized in very long pieces Lagging strand 0 d makes RNA first then DNA 5 3 0 will end up running into leading strand of other fork 6 binds and goes until it runs in another primer by d 0 primer must be removed FEN 1 in normal replication exonuclease activity5 3 0 New DNA must replace primer 0 6 keeps going extends Okazaki fragment to end of leading strand 0 DNA made by d not proofreaded o FEN 1 removes all DNA made by d 6 can keep going and fill in gaps while proofreading Ligase connects Okazaki fragments with phosphodiester bonds FEN 1 igase and 6 come in a package with a clamp Clamp oader realigns to proper position 0 Prevents premature dissociation of enzymes to high processivity high efficiency Loadingbinding process is cumbersome and slow I Once enzymes get goingfast Domain 1 folded area in 3 protein structure Subunit 2 folded polypeptide in a 4 protein structure 6 bound to Z 0 DNA accommodates enzyme 0 Lagging strand oops around Replisome Set of enzymes at one fork helicase DNA pol Topoisomerase not included E Coli DNA Pol 3 o 5 3 DNA polymerase activity 0 high processivity I B clamp o 3 5 exonuclease activity extremely activity Repair 0 Other enzymes will come back through DNA and x errors after replisome goes through New chromosomes are getting shorter Cells at rst are dividing quickly 0 In time they lose the ability to divide Chromosomes are getting shorter and shorter Any more replication would cause chromosome to shorten to a dangerous point Important genetic information will be lost Any further replication is prevented Seen in cell culture 0 Skin cells easily seen necessary for wound healing Younger quicker to heal Older longer to heal Cell division is needed to renew and heal Aging is not purely a telomere issue but telomere research is major in aging work DNA Pol alpha didn t put the primer all the way on the end of the strand 0 Structurally cannot do it o Creates gap in new strand 0 Even if primer was there and could be removed new nucleotides can t be added at 5 end 0 Thus shorter and shorter strands result Chromosome is shortened at both ends 0 Cumulatively over time o Lagging l leading antiparallel o Telomerase is adding to the 3 end at both ends Telomerase A special DNA Polymerase Telomerase adds new nucleotides to the 3 longer end of the template How does it add new DNA when there is no template 0 Telomerase has its own template o It is part protein and part RNA RNA component is this internal template Usable part of template is very small 68 nucleotides Part of the telomerase template is complementary to 3 end of template strand Adds new nucleotides and slides down repositions and adds more nucleotides 0 Does this multiple times to extend the end 0 Not an action that is exactly timed with replication There will be many rounds of replication where chromosomes are getting too short a Activates telomerase to get chromosomes back to proper length Mechanism that counteracts shortening so that loss does not do too much damage 0 The DNA that is added is like a buffersacri cial o Meant to be lost 0 Junk DNA still has function not coding though Repetitive noncoding sequence o Is working off of one short template Can only add that one short sequence over and over again 0 Bigger gap in new strand results 0 Space for new Okazaki fragment 0 Strands are still not equal in length Not same problem Chromosomes have been lengthened with DNA at both ends of chromosome Cumulative loss has been balanced and offset When we get older it seems like telomerase is not doing its job anymore Telomerase present amp active in o Germ cells O O Not the gametes but the diploid cells in ovaries amp testes that produce the gametes CancerceHs Unicellular eukaryotes Necessary because new cells are offspring Telomerase absent in O O Somatic cells Prokaryotes Chromosomes are circular problem never occurs 0 Practically all cells have the same DNA 0 Expression of different genes determines the differences in cells 0 Telomerase genes have been shut off in somatic cells Central dogma of genetics Crick had idea of DNARNAprotein O O O 0 DNA is template upon which RNA is made transcription RNA Polymerase RNA is template upon which protein is made translation Ribosome Many kinds of RNA transcribed from DNA ON LY mRNA ARE TRANSLATED Info does not ow right to left but ows only left to right There are exceptions however a Telomerase DNADRNA DNA nucleotides are coming out of solution and lining them up with their internal RNA template 0 In the class of viral enzymes a Reverse transcriptase DNADRNA 0 Viral enzyme found in retroviruses ie HIV Proteins Function 0 Enzymes o Regulators o Receptors 0 Structural 0 Transport 0 tRNA 0 transport function 0 rRNA o structural function 0 snRNA o catalytic some proteins have protein like functions 0 don t need to be translated into proteins Gene 0 Codes for a protein or a noncoding RNA 0 Can contrast genes and nongene sequences with transcription 0 Genes capacity for transcription I Must have promoter n 3 functions that all occur to get transcription started 0 provide a site for RNA Pol binding 0 provide a site for initial unwinding breaking of H bonds l strand separation 0 mark the exact start point of the gene exact nucleotide where transcription begins 0 Nongene sequences no transcription Elongation Happens after rst few nucleotides 0 What marks elongation is when RNA starts moving 0 RNA Pol has helicase activity 0 DNA is closing up behind RNA Pol 0 Only strand as template template strand 0 Other strand is sense strand 0 Similar to RNA strand except thymine amp uracil No proofreading or checking for errors in RNA 0 RNA Pol moves downstream o Came from upstream 0 First nucleotide transcribed is 1 As you count downstream you get more positive 2 3 4 etc As you count upstream you get more negative 1 2 3 etc Conserved sequences Promoter is entire sequence 0 Necessary for basic level of transcription Sequences within the promoter Islands of important sequences that are conserved o If you look from species to species you will nd similar sequences in promoters Individually one sequence cannot initiate trxn but together they can with right spacing for some quantity of trxn Not only for trxn o DnaA box amp AT rich regions replication Conservation function How can you communicate what the sequence looks like if there is no one sequence that exactly describes it l Consensus sequence One exact sequence that isn t exactly what the conserved sequence looks like 0 Still a good description of what it typically looks like Particular regulatory sequence with identi ed relative position to several different genes that have the same function Collecting samples of conserved sequence 0 GCTGAT o TCCACT o GCTAAT o GCGAGT o GCTGTC Derive a consensus agreement I Position by position I GCTRNT In a conserved sequence not all positions are equally conserved o No one nucleotide is responsible for the entire function 0 C second position is clearly the most important May not do all the function but may be required for the funcUon Sometimes it s not the chemistry of the individual base but the shape that is important to the sequence 0 One shape is a lot more common than the other shape Purine vs pyrimidine n In example above fourth position is all purines PurineRPu PyrimidineYPy Any nucleotideN o Unconserved nucleotide within a conserved sequence 0 Why DNA binding domain of regulatory sequence I Might not make contact at every base pair a 5th nucleotide doesn t really matter 0 free to mutate no effect on function Prokaryotic promoters Know name of sequences 10 or 35 where it is located eu vs pro which strand it s on how it functions 0 Don t need to know speci c nucleotides Consensus sequences are derived for the sense strand 0 When you look up on the table you need to know which strand it s on o How are they named o How are locations derived Upstream of 1 gene Which nucleotide is the 10 in the 10 sequence a Downstream middle nucleotide o If there is an even number as long as it s in the middle it s okay Initiationfunction of promoters 1 Provide a site for RNA Polymerase binding 0 35 sequence 0 RNA Pol not just lining up on only 35 sequence RNA Polymerase core enzyme 0 Alpha alpha beta beta prime omega subunits Sufficient for polymerizationmaking RNA 0 Cannot nd promoter although it binds to DNA RNA Polymerase holoenzyme Core enzyme with sigma subunitfactor Bind promoters speci cally Covers entire promoter 0 Sigma within the holoenzyme recognizes primarily the 35 sequence in the promoter 2 Provide location for initial unwindingopening up of helix 0 10 sequence 0 AT rich Easily meltable fewer H bonds Just any AT region alone does not make 10 sequence Some 10 sequences have 6 5 and C s Unwinding spreads to include 1 3 Mark exact location of where trxn will start 0 up to the rst ten nucleotides is still rst step of initiation 0 makes sure trxn gets off to good start 0 10 sequence is not really 10 nucleotides upstream same with 35 o trxn initiates 35 nucleotides downstream of the site where sigma recognizes o trxn initiates 10 nucleotides downstream from where unwinding starts RNA Polymerase binds and does not move along the RNA 0 All initiation takes place with RNA in position 0 When trxn initiation is completed then RNA Pol will move downstream o Elongation Helicase activity needs to be centered over the 10 sequence 0 For unwinding 25 nucleotide distance is between the center of the helicase activity and the sigma factor domain Elongation After rst 10 of RNA are polymerize sigma factor dissociates 0 Sigma factor is sequence speci c 0 Core enzyme can move 5 3 down the gene 0 Another protein binds to the core enzyme in sigma factor s place 0 NusA Function is to slow down the core enzyme I Binds to core enzyme for elongation I Not present during trxn initiation Termination 1 Rho dependent termination o Rho binding site Cannot bind prematurely 0 Only has binding site when trxn has been successful 0 Rho cannot bind to DNA can only bind to mRNA 0 Rho moves 5 3 as soon as it binds O Catches up to RNA Pol n Rho has helicase activity Unwinds and breaks H bonds 0 Separate RNA and DNA 0 Melts RNA off of DNA template 0 Also thought to help RNA Pol dissociate 2 Rho independent termination more common in ecoi stem oop hairpin 0 needs to be at least 8 nucleotides on rst side 0 minimum of 5 nucleotides in top loop portion 0 as soon as this speci c sequence comes out of RNA Pol it will fold on its own occurs very quickly and snaps together yanking on the ends I end with the lesser mass is yanked more a enough to exert force and pull on H bonds in easily meltable region AU rich region 0 all those uracis will be popped off and mRNA will oat away 0 RNA Polymerase has no 3 end anymore 0 Dissociates from DNA mRNA trxn in eukaryotes 0 larger promoter more sequences 0 positions are less consistent 0 sequences named for their consensus sequences not location locations are more exible except TATA box which is usually 25 region 0 look at entire consensus and nd strong similarities to names 1 Provide a site for RNA Polymerase binding 0 RNA Polymerase doesn t bind to any of the sequences 0 Transcription factors bind instead 0 RNA Pol bind onto the group of trxn factors just one of them Multiple trxn trxn initiation complex 2 Provide a site for unwinding to begin 0 TATA box 0 AT rich Easily meltable Closer to 1 3 Mark exact start point of trxn 1 is 1 because of spatial relationship between 25 sequence TATA box 0 Not enough conserved sequences at 1 site to stand as marking the spot of trxn initiation RNA Polymerases in eukaryotes o RNAP l o Transcribes most of the rRNAs not the smallest one 55 0 Two trxn factors Binding sites are core element and upstream control element 0 RNAP II o Transcribes mRNAs o TFIIA TFIIB TFIID Trxn factors that bind to particular conserved sequenced and form a complex together Binding sites at protein encoding genes a Not all protein encoding genes have all the sequences 0 One can be swapped out 0 Ex 2 GC boxes or 2 CAAT boxes 0 RNAP Ill 0 Transcribes small RNAs eg tRNA 55 rRNA etc o TFlllA TFlllB TFIIIC 1550 conserved sequences are completely different 0 How are RNAP speci c for these certain genes 0 Trxn factors are different for each sequence 0 Each RNAP has its own dedicated set of trxn factors Named in a way that makes that clear Elongation in eukaryotes 0 RNA Pol will change shape during initiation but will not move down the gene until elongation starts 0 Some trxn factors go with RNA Pol some stay behind I Some trxn are functional for elongation some only for initiation Other elements of elongation are same as prokaryotes except sigma 2300 Termination in eukaryotes Different termination sequences for each of the RNA Polymerases See RNA processing notes Prokaryotes 0 Multiple protein encoding genes will often share a promoter 0 Makes expression easier Might all be necessary for the same process not necessarily same function 0 Only one 1 0 One transcript for all these genes 0 Polycistronic mRNA many genes 0 Each polypeptide has to have its own start codon o Ribosome gets to only the stop codon releases the completed polypeptide and a new ribosome will initiate translation all over again for the new polypeptide at a new start codon o No reason to separate them 2900 0 Post transcriptional modi cationRNA processing 0 None in prokaryotes o Prokaryotic mRNAs are not processed o Trxn and translation are coupled in prokaryotes rRNAs 0 multiple genes are sharing a promoter one transcript not translated a one 1 I one trxn terminator different rRNAs that need to go assemble in a ribosome I cannot stay together a need to separated 0 different destinations Endonuclease needed to cut up long transcript 3300 a Creates shorter fragments of RNA 0 Rare bases in tRNA Dihydrouracil modi cation of uracil Thymine modi cation of uracil Pseudouracil modi cation of uracil Inosine I Not just for tRNAs a Rare bases are common in tRNAs but rare in other RNAs Editing enzymes made in post translation a Different editing enzymes for different rare bases amp different locations on transfer RNA 0 Shape of tRNA is important for enzymes to recognize n tRNAs are heavily edited 0 Most tRNAs are transcribed with other tRNAs 0 Share a promoter 0 If one is transcribed all of them are transcribed 0 One transcript o Endonuclease cleavage 0 RNA editingrare bases Eukaryotes Each protein encoding gene has its own promoter 0 Flexibility o Easier to regulate the genes independently 0 Not about simplicity but about control 0 Pre mRNA go through processing before becoming mature mRNA 0 1 5 cap attached 0 2 PolyA tai attached 0 3 Splicing rRNA 0 break the eukaryotic rule I only one promoter n every rRNA has one copy of 18S rRNA 585 rRNA 28 rRNA prevents waste by clustering them under the control of one promoter equal production 0 last part of promoter rst part of transcribed region 0 promoter overlaps rst gene 4800 0 tRNA 0 promoters are internal in the gene 0 RNA Pol binds on and overlaps the 1 4850 Different class of genes a RNA Pol 3 0 Each gene has its own internal promoter Regulated and transcribed independently Separate transcripts 0 RNA editingrare bases mRNA processing 0 1 5 cap 0 cap itself is a guanine nucleotide not a regular guanosine it s a methylated guanosine 0 attachment is weird 3 phosphates instead of one attached backwards a super weird bond n 5 C l 5 C o 1 The cap and tail protect the ends of the mRNA from being attacked by exonucleases 5 3 for cap 3 5 for tail 0 regular 2 min half life would never work 0 cap and tail buy time totally unrecognizable to normal nucleases no degradation 0 2 Binding site for ribosomes o ribosome will have to recognize the mRNA to initiate translation 0 not the site of initiation but is the binding site 0 2 PolyA tai binding site for protein that speci cally recognizes long stretches of adenosines a multiple copies of the protein bind along the tail a protein is bound on the tail on mRNA occupying thatspace 0 poly A binding protein a dif cult for an exonuclease to grab on and cut the 3 end a proteins are there to keep the tail from being shortened not as good as cap but dramatically slows it down 0 some have halflives of days to weeks I Poly adenylation signal a 5 AAUAAA 3 a binding site for 2 proteins endonuclease 10200 0 breaks phosphodiester bonds 3 end is created poly A polymerase 0 adds nucleotides to 3 end adenosine 15200 m Endonuclease and poly A polymerase work together RNAP ll continues transcribing for thousands of nucleotides n Stops transcribing and releases second part of transcript a No particular distance or sequence that triggers termination Second part of transcript is immediately degraded o 3 Splicing in vertebrates there are 78 exons with huge variations exons are typically small large amount of intron material that is removed discarded and degraded exons go back together in the exact same order n doesn t mean they are spliced in that order n splicing must be done very exactly a 3 conserved sequences in introns 2 of them determine splice sitejunctions 0 long sequences that exactly mark those sites o GU and AG are highly conserved across all eukaryotes Only most conserved nt out of longersequences At 5 amp 3 ends of introns Branch point conserved sequence Spliceosome El Small nuclear RNA snRNA o snRNA U1 U2 U4 U5 U6 Small proteins that bind with each of the snRNAs to make small nuclear ribonucleoproteins snRNPs Ribonucleoproteins 0 Nothing to do with ribosome o Ribo ribose o snRNP U1 U2 U4 U5 U6 0 all of those together spliceosome snRNPs have to assemble on the intron snRNP U1 binds at GU snRNP U2 binds to branch point U1 and U2 bind to each other Bendingfolding the RNA Brings the two exons that are going to be joined in proximity to each other First cut comes at the 5 splice site right under the G 955 lntron is not a circle but a lariat lasso n Covalent phosphodiester bond between the 5 G amp 2 A in branch point 0 Forms the lariat n Joins the exons together after splicing the introns a All introns in eukaryotic mRNAs in the nucleus are spliceosome type introns 0 Reference lecture outline front page gure a Process of splicing must be repeated for other exonsintrons Alternative splicing Few genes l many polypeptides Limited to inclusion vs exclusion of the exons 0 Are all the exons going to be present in the nal mRNA 0 Or is one or multiple exons going to be spliced out o No reordering Prokaryotes 2655 0 Have introns too Bacterial tRNA can have introns No spliceosomes in prokaryotes lntron folding in on itself 0 Brings exons closer together 0 Creates a different shape Really intricate folding in the intron 0 Gains function Ability to splice itself out Also joins together exons Ribozyme a RNA amp catalyst Self splicing introns can be found in mitochondria chloroplasts protists and bacterial tRNA Translation 3200 Begins at start codon and ends at stop codon Codon 0 Consecutive and nonoverlapping o Triplet of RNA nucleotides that correspond to one amino acid in a polypeptide 0 Stop codon corresponds to no amino acid in polypeptide No corresponding tRNA Codon in mRNA base pairs with complementary anticodon in tRNA 0 Codons are also in the sense strand of DNA 0 Start codon 0 Sets the reading frame Grouping of nucleotides into the codon Codons are consecutive and nonoverlapping Same string of RNA can give multiple reading frames depending on where you start 0 Provides place where ribosomes start translating 0 Not exactly at the 5 end 1 nucleotide is at the 5 end 1 amp start codon are not the same and stop codon amp trxn termination sequence are not the same acid 0 separate function 0 separate location 0 separate sequence 0 3 UTR amp 5 UTR exists because of this separation tRNA are set into order by codons amino acids are set into order by tRNAs Every amino acid 0 Central carbon with 4 diff group 0 Amino o H o R group side chain 0 Carboxyl Carboxyl end of polypeptide is attached to amine group of the new amino acid 0 Arrangement of the varying functional groups on the different side chains that determines the folding and function what it can bind to Carboxyl end of polypeptide attached to amino group of the new amino Polypeptides are made aminocarboxy Corresponds to 5 3 on the mRNA Nformyl methionine Only found in bacteria Cannot be added to a chain Only exists in the rst position of the polypeptide Start codon codes for Nf met in bacteria tRNA 3 acceptor stem 0 Amino acid is covalently attached in ribosome 4800 Boomerang shape of tRNA 0 TLIJC arm has folded over and up so that it s on top of the D arm Binding to each other with weak bonds Tertiary structure 0 Weak bonds between bases in tRNA that would contribute to the shape 0 Rare bases that are all over tRNA have a lot of opportunity to tweak the tertiary structure of tRNA Increase the speed and accuracy of translation 0 Genetic Code Triplets always listed 5 3 Triplets are all codons NOT anticodons 0 mRNA or sense strand of DNA Don t worry about one letter abbreviations of amino acids Three letter abbreviations are useful 0 Can leave it abbreviated 0 AUG is start codon and encodes methionine 0 Can code for methionine and not start codon o If coded for start codon aso coded for methionine Except nf met in bacteria Coding is universal translation side 0 How cloning works between human DNA into bacteria 0 Exception is protists and mitochondria Not whole code just bits 0 Code is degenerate o Redundant More than one codon corresponding to most of the amino acids 0 Different ways to encode but not ambiguous Any given codon only codes for one amino acid Wobble 10045 0 G is paired to U o G swung down and shifted over to the right 0 Not lined up to the U in same way 0 Partial neg lined with partial pos and vice versa 0 G amp U wobbled to nd an alignment where opposite partial charges are next to each other 0 Some bases in codon amp anticodon in RNA have more room to move than bases in DNA GU pairings work in either orientation o I can pair with C U if offset and A a little wobble Wobble rules 0 All Watson and Crick base pairing rules apply 0 GU In the third position 0 lanything but guanine In the third position Ribosomes Composed of ribosomal subunits o Composed of rRNA amp protein 0 Function is to make proteins 0 Structure amp function is a chicken amp egg problem 350 o 2 daughter cells inherit lots of ribosomes I lots of different proteins I include proteins that encode for ribosomal proteins 0 bulk of ribosome is 23 RNA and 13 protein folding of RNA is what created the structure of the ribosome n accommodates the function proteins are too few and small to do this 0 rRNA is what carries out basic functions of the ribosome NOT the proteins 0 RNA came rst ancestral ribosomes were completely RNA Proteins came later a Necessary to ll holes a lncrease speed amp accuracy of translation Prokaryotic translation 0 lnitiation 0 Initial assembly of the parts 0 Finitiation factor Assistinghelping the steps Not the primary molecule carrying out the steps Aid in initiation and dissociate after a Different from rRNA which are integral parts of the ribosome and remain o Shine Dalgarno sequence Upstream of the start codon Untranslated 5 region AGGAGG7 ntAUG n The way the 30S binds to mRNA is by base pairing n Shine Dalgarno sequence of mRNA base pairing to complementary sequence in 16 0 Allows the small subunit to bind to mRNA Which AUG is the start codon n The one that has the speci c spatial relationship to Shine Dalgarno sequence a Whatever is 7 nt down gets plopped into a spot on the 305 0 Spot where rst tRNA comes 0 P site a Sounds like transcription initiation with the promoter 0 tRNA brings rst AA already attachedcharged to P site if tRNA not charged they won t be in the right shape sti boomerang shape but not enough anticodons are bent out of shape cannot bind to ribosome o tRNA its AA IF2 amp GTP like ATP form a complex that has the right shape to bind on to the ribosome only tRNA that binds to the P site 0 these molecules are not just oating around and come together randomly works instead in a series of speci c ordered steps when molecules bind together their shape changesljfunction is altered each event like binding triggers the next event ribosome does not have master plan I molecules are just triggering step after step 0 GTP is part of the shape change function change each step triggering the next story 0 Not about energy but about control 0 SOS binds and IF dissociates GTP mediates this It s cleaved o Elongation Ribosome is complete 705 and ready for elongation o How each subsequent AA is added 0 Space open for the next tRNA complex to come in o A site is for aminoacyl site Where new AAs are brought Charged tRNA EFTu elongation factorTu amp GTP form a complex that comes to this site a When this complex comes in EFTu doesn t actually t in because too big o tRNA is bent over and EFTu is sticking out 0 AA is actually far away from the polypeptide in P site 0 Peptide bond cannot be formed 0 Who knows if it s the right tRNA and subsequently the right AA 2500 tRNA binds but EFTu blocks peptide bond n H bonds btwn codon and anticodons o Pulling down on the tRNA amp deforming it a little bit 0 Lots of H bondsgood match 0 When tRNA shape changes EFTu cleaves GTP n EFTu changes shape again a No longer has af nity and is bound to GDP instead a EFTu dissociates tRNA pops into place a AA is now right next to the peptide Peptide bond forms 0 P site is for peptidyl site However much the polypeptide is goes there No enzyme is catalyzing the peptide bond n Named in anticipation Peptidyl transferase a No such enzyme though a Actually an activity of the 23S rRNA ribozyme Ribozyme is an RNA with catalytic ability not the ribosome n Transfers the growing polypeptide onto the new AA that s attached on the tRNA in the A site a Cuts the peptide off its tRNA n Makes new bond between peptide and new AA n Events are coupled in one reaction a Energy in the covalent bond that is broken powers the new peptide bond EFG plays central role in translocation n 5 3 movement of the ribosome by one codon or 3 nt down the mRNA tRNA is in relative same position it is the ribosome that moves a tRNA complex is moved to P site 0 still on same position on the mRNA n A site is now open for the next tRNA complex Cycle repeats over and over again until the stop codon Termination o How stop codons work 0 Stop codon is now in A site 0 There is no tRNA that recognizes the stop codon RF1 release factor 1 recognizes the stop codon instead a Shaped like a boomerang just like a tRNA 0 Based on crystal structure that is extracted by xray diffraction that is still used today Rosalind Franklin amp xray diffraction o Striking similarity 0 Not similar internally tRNA made out of RNA RF1 made out of proteins 0 mimicry n exhibits molecular mimicry RF3 is lling the same role of EFTuIF 2 to complete the full shape of the complex I RF1 has series of amino acids that have H bonds with the stop codon a When those H bonds form that triggers RF1 s catalytic activity Cuts the completed polypeptide off the tRNA in P site 0 RF1 not actually cutting it off 0 Tricks peptidyl transferase into cutting the polypeptide off 3 stop codons n UAG UAA RF1 amp RF3 complex a UAA UGA RF2 amp RF3 complex RF2 almost identical to RF1 in terms of shape a Modi cation at the bottom to recognize a different stop codon 0 Once polypeptide is released all the parts will dissociate and disassemble RF3 helps the subunit to repel n Causes all other parts to come apart Eukaryotic translation 0 See comparecontrast chart Mendel 1860 s differences in the genotype allelesdifferences in phenotype Garrod 1902 0 MD who had some patients exhibiting a strange phenomenon where their urine was black alkaponuria o All relatives of each other Urine is by product of metabolism 0 Differences in urinedifferences in metabolism 0 Differences in genotypeljdifferences in metabolismljdifferences in phenotype Certain diseases are inborn errors of metabolism Beadle amp Tatum 1941 0 Differences in genotypeljdifferences in enzymesljdifferences in metabolismljdifferences in phenotype o If enzyme is nonfunctional that causes the difference in the metabolism Will block one of those metabolic pathways 0 Enzymes catalyze steps in the metabolic pathways 0 One gene one enzyme hypothesis 0 One gene encodes one enzyme Does not apply to all genes In Alternative splicing one gene can encode multiple polypeptidesenzymes n One gene encodes no enzymes tRNAs rRNAs snRNAs etc a Genes can encode different subunits of a protein s quaternary structure 800 n Genes for proteins that are not enzymes transport protein hemoglobin antibodies receptors etc 0 Lots of limitations 0 One gene one polypeptide 0 Each gene encodes one polypeptide o Addressed the last two exceptions above 0 Two exceptions are still left Alternative splicing One gene zero polypeptides Biochemical pathway 0 Series of steps where chemicals go through stepwise chemical modi cations 0 Catalyzed by enzymes PKU 0 Combo of neurological 0 Sam the genetics graduate student notices that a mutant strain of yeast a fungus not prokaryote but a microscopic eukaryote contains an mRNA that is longer than the corresponding mRNA in the nonmutant strain Sam was surprised to discover that the amino acid sequence of the polypeptide was identical in the mutant and nonmutant strains Where is the mutation most likely located 0 Could be on the 5 3 untranslated region Spacing doesn t matter because of scanning for Kozak sequence in eukaryotes lntron No that s on the premRNA In the GC box In the stop codon At a splice junction In the polyadenylation signal PolyA polymerase amp an endonuclease binds here at the 3 end of the premRNA after the stop codon 3 UTR 1200 A3 chapter 13 tRNAs should not be able to recognize stop codons or multiple proteins eg both leucine and phenylaline P5 old exam 4200 0 identify any known conserved sequences 0 ex AT rich region for 10 sequence or TATA box identify a 35 sequence to determine which one it is 0 start transcribing from the 1 same as sense strand except UT downstream from 1 on sense strand 0 start codon is contained in the Kozak sequence BDNA amp ZDNA o ZDNA forms from BDNA right behind RNA Polymerase FEN1 Used in replication normal and non and repair 0 In normal replication it has 5 3 exonuclease activity T5 old exam 0 a Telomerase only creates a repetitive speci c noncoding sequence from its own template RNA cTeomerase will add a new nucleotide to the very end of the 3 end of the longer strand 1 old exam 0 you cannot apply Chargaff s rule to singlestranded molecules like RNA 0 or you could say there is approximately 15 because the quantity of cytosine is roughly similar amp not all of DNA is transcribed goom o
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